We have previously shown that cinnamoyl derivatives of 14β-amino-17-cyclopropylmethyl-7,8-dihydronormorphinone and 7α-aminomethyl-6,14-endoethanonororipavine have pronounced pseudoirreversible μ opioid receptor (MOR) antagonism. The present communication describes the synthesis and evaluation of fumaroylamino analogues of these cinnamoylamino derivatives together with some related fumaroyl derivatives. The predominant activity of the new ligands was MOR antagonism. The fumaroylamino analogues (2a, 5a) of the pseudoirreversible antagonist cinnamoylamino morphinones and oripavines (2b, 5b) were themselves irreversible antagonists in vivo. However the fumaroylamino derivatives had significantly higher MOR efficacy than the cinnamoylamino derivatives in mouse antinociceptive tests. Comparison of 2a and 5a with the prototypic fumaroylamino opioid β-FNA (1a) shows that they have similar MOR irreversible antagonist actions but differ in the nature of their opioid receptor agonist effects; 2a is a predominant MOR agonist and 5a shows no opioid receptor selectivity, whereas the agonist effect of β-FNA is clearly κ opioid receptor (KOR) mediated.

Investigation of the pharmacology associated
with the individual
opioid receptors, μ (MOR), κ (KOR), and δ (DOR),
has been majorly advanced by the availability of antagonists selective
for each of them. For MOR, one of the most well used antagonists has
been β-FNA (1a, Chart 1),1 which owes its selectivity to the presence of
the fumaroylamino group preferentially interacting covalently as a
Michael acceptor with the amino group of Lys233 in the MOR.2 β-FNA also has KOR agonist activity of
short duration.1 Our interest in this field
has been primarily in epoxymorphinan structures with cinnamoylamino
substituents.3−7 Though the 6β-cinnamoylamino analogue (1b) of 1a had predominantly KOR agonist activity in vivo,8 the p-chloro- and p-methylcinnamoylamino derivatives (1c, 1d) had a profile more similar to that of 1a.9

In contrast the 14-cinnamoylaminodihydromorphinones
clocinnamox
(C-CAM, 2c) and methcinnamox (M-CAM, 2d)
had no significant opioid receptor agonist activity in vitro or in
vivo but were MOR-selective antagonists of greater potency and longer
duration than 1a.7 Though
there was no evidence of covalent binding to MOR, 2c and 2d were able to cause long-term inhibition of MOR more effectively
than 1a and have been categorized as pseudoirreversible
MOR antagonists.7,10 The oripavine-related cinnamoylaminomethyl
derivative 5b has an opioid receptor profile similar
to those of 2c and 2d.6,11

One of our particular aims in this field has been to discover compounds
with a profile not dissimilar to that of the opiate abuse treatment
agent buprenorphine.12 Buprenorphine is
a partial agonist at MOR with a long duration of action. When the
agonist action is blunted, which occurs following repeated dosing
when tolerance has developed, buprenorphine becomes a pseudoirreversible
antagonist13 that can block the actions
of subsequently administered opiates. In addition to this activity
at the MOR, buprenorphine is an antagonist at KOR and DOR. There has
recently been interest in a combination of buprenorphine with sufficient
naltrexone to essentially eliminate the MOR partial agonist effect,
creating a functional MOR/KOR/DOR antagonist.14 This combination could be used to prevent relapse in recovering
opiate addicts. Since the cinnamoylamino (2) derivatives
had also shown similar irreversible MOR antagonist characteristics
compared to buprenorphine and also bound to KOR and DOR, it was of
interest to determine what effect replacement of the cinnamoylamino
group by a fumaroylamino moiety would have on their activity and whether
ligands with profiles of interest for the treatment of drug abuse,
or prevention of relapse to drug taking, could be obtained. Significant
similarities between the series have been found, while some notable
differences in pharmacological profile were also noted.

Synthesis

2a, 4a, and 5a were prepared
by acylation of the known primary amines (8, 11a, 11b)3,6 with methyl (3-chloroformyl)acrylate
(Schemes 1 and 3), while
hydrogenation of 2a yielded 6 in respectable
yield (Scheme 1). Acylation of TBDMS-protected
naltrexone (9) with methylfumaroyl anhydride followed
by removal of the protecting group allowed access to 3a (Scheme 2).

Results

Opioid receptor binding studies were conducted
in Hartley guinea
pig brain membranes in which the displaced radioligands were [3H]DAMGO (MOR), [3H]Cl-DPDPE (DOR), and [3H]U69593 (KOR) using previously reported procedures.14 The results are shown in Table 1; the four fumaroyl derivatives (2a, 3a, 4a, 5a) and one dihydrofumaroyl derivative
(6) all showed high affinity for all three opioid receptors,
although 4a had lower affinity at MOR than the other
ligands, while 3a had lower affinity at DOR and had a
binding profile quite similar to that of 1a.

Functional opioid receptor activity was determined
in vitro in
electrically stimulated isolated tissue bioassays.14 In the guinea pig ileum (GPI) which has populations of
MOR and KOR but not DOR, the new ligands were either partial agonists
(2a, 3a, 5a) or had no opioid
receptor agonist activity (4a, 6) (data
not shown). The partial agonist activity of 2a showed
the highest level of efficacy (54% inhibition of the electrically
stimulated twitch) which was inhibited by the selective MOR antagonist
CTAP.15 The partial agonist activity of 3a (30–40% of maximum) was prevented by norBNI16 but not by CTAP, indicating that it was KOR
mediated. The partial agonist activity of 5a (maximum
36% at 40 nM) was not prevented by CTAP or norBNI, which may suggest
it is of irreversible character.

The mouse vas deferens (MVD)
expresses populations of all three
opioid receptors but is most sensitive to DOR agonist activity. None
of the new ligands showed agonist activity in this assay, but they
all displayed potent antagonist activity against selective agonists
for MOR (DAMGO), DOR (DPDPE), and KOR (U69593) (Table 2). All the tested new ligands (3a, 4a, 5a, 6) were potent MOR antagonists with
subnanomolar Ke values. They were only
slightly less potent KOR antagonists but significantly less potent
(10- to 100-fold) DOR antagonists. Whereas there was little difference
between binding affinities (Ki) and antagonist
potencies (Ke) for the 7α-aminooripavine
derivative (4a), for the 7α-aminomethyl derivative
(5a) MOR and KOR antagonist potencies were very much
higher than MOR and KOR binding affinities. This indicates that 5a binds much more tightly to opioid receptors under physiological
conditions than 4a.

The new ligands were evaluated in mouse antinociceptive
tests using
thermal (warm water tail withdrawal, TW) and chemical (acetic acid
induced writhing, AW) stimuli using procedures reported previously.7 In TW, only 2a showed any antinociceptive
activity. In the 50 °C TW assay it had substantial
agonist activity, reaching 60% of the maximum effect at the highest
dose tested (100 mg/kg) (Figure 1) with about
half the potency of morphine. With water at 55 °C, 2a had reduced agonist effect, giving 30% response at 100 mg/kg, whereas
the same dose of morphine showed 100% response.

Given our interest in compounds that display initial
agonist activity
followed by antagonism and in view of its substantial agonist effect,
the antagonist effect of 2a on the morphine dose–response
curve using 55 °C water was measured 24 h after 2a was administered. At a dose of 10 mg/kg 2a, the morphine
curve was shifted 6-fold to the right, whereas a dose of 100 mg/kg 2a totally flattened the morphine response (Figure 2). At a shorter time point (30 min), the 10 mg/kg
dose had less effect than at 24 h, suggesting a slow onset of antagonist
activity (data not shown). The higher dose had very long duration,
remaining effective for at least 9 days (Figure 3). The morphine dose–response curve in TW was also substantially
flattened by doses of 32 mg/kg 3a and 5a administered 30 min before morphine, whereas the effect of the same
dose of 6 and 4a was to shift the morphine
curve 10-fold and 30-fold, respectively, to the right in essentially
parallel fashion. When 3a, 4a, and 6 were administered with 24 h pretreatment, the morphine dose–response
curve in each case was shifted about 4-fold to the right, whereas
in an equivalent experiment 5a produced a 30-fold rightward
shift. Thus, as MOR antagonists, 2a and 5a are both of long duration and appear to have irreversible characteristics
whereas 3a, 4a, and 6 are less
impressive as irreversible MOR antagonists and may be essentially
reversible, as predicted by the in vitro data where the Ki in binding was the same as the Ke in the functional assays for 3a, 4a, and 6, indicating reversible binding.

Although 3a, 4a, 5a, and 6 had no antinociceptive activity in
TW, 5a and 6 as well as 2a had
significant activity in AW. 2a at a dose of 10 mg/kg
inhibited writhing to 90% of the
maximum possible inhibition, as did 6 at 32 mg/kg; 5a at the same dose showed 80% inhibition. By use of selective
antagonists for the individual opioid receptors, the antinociceptive
effect of 2a in AW was shown to be predominantly MOR-mediated,
with some DOR involvement, whereas that due to 5a had
significant contributions from all three opioid receptors. This is
illustrated in Figure 4 for 2a. 3a and 4a had no antinociceptive activity
in AW; in the case of 3a this was in contrast to partial
agonist activity in vitro in GPI. However, the latter was of low efficacy
and mediated by KOR agonism.

The opioid receptor antagonist profile of the new
ligands was also
investigated in vivo in the AW assay. All the ligands tested (2a, 3a, 4a, 5a) had
opioid receptor antagonist activity when administered at a dose of
32 mg/kg, 24 h before determination of the effect of ED100 doses of selective opioid receptor agonists for MOR (morphine),
KOR (bremazocine), and DOR (BW373-U86) (Table 3). In this assay the three fumaroylamino derivatives (2a, 4a, 5a) showed preference for MOR antagonism
whereas the naltrexone derivative (3a), which had overall
the least opioid receptor antagonist activity, had no selectivity.

The 14-fumaroylaminomorphinone derivative (2a) was
evaluated in the morphine-dependent rhesus monkey model.17 In withdrawn monkeys, 2a at doses
of 0.1 and 0.5 mg/kg did not substitute for morphine or attenuate
withdrawal (data not shown) but in nonwithdrawn monkeys at a dose
of 2.0 mg/kg it precipitated withdrawal symptoms that lasted longer
than those produced by a standard dose of naloxone (0.05 mg/kg) (Figure 5).

Discussion and Structure–Activity Relationships

We showed that replacement of the carbomethoxy group of (1a) with aryl groups gave new 6β-cinnamoylamino ligands
(1b–e) of which the p-chloro and p-methyl derivatives had profiles similar
to that of 1a(9) and the unsubstituted
cinnamoylamino and p-nitro analogues had predominant
KOR agonist activity.8 These relationships
of cinnamoylamino to fumaroylamino derivatives motivated the present
investigation of fumaroylamino derivatives (2a, 4a, 5a) related to our previously reported cinnamoylamino
derivatives (2b–e, 4b–e, 5b–e).4−6

In the series of 14-cinnamoylamino-7,8-dihydromorphinones
(2b–e) the dominant in vitro and
in vivo
activity was MOR antagonism.7,17 In the current investigation
it has been shown that the equivalent fumaroylamino derivative (2a) is a MOR partial agonist because in TW it had substantial
MOR efficacy, but it also developed a long-lasting morphine antagonism
having irreversible characteristics. In this respect it most closely
resembled the p-nitrocinnamoylamino derivative (2e), the only member of the 14-cinnamoylaminodihydromorphinone
series to have substantial agonist and MOR antagonist activity in
TW.5 Next most similar to 2a among the 14-cinnamoylamino derivatives was the unsubstituted cinnamoylamino
derivative (2b), although the latter was somewhat less
effective than 2a as a pseudoirreversible MOR antagonist
and as a MOR agonist in vivo.52c and 2d, the chloro- and methylcinnamoylamino derivatives,
are quite different from 2a in having no opioid receptor
agonist activity.

The 7α-fumaroylaminomethyl oripavine
derivative (5a) had a profile similar to that of the
14-fumaroylaminodihydromorphinone
derivative (2a), although the antinociceptive efficacy
and the duration of in vivo MOR antagonism by 5a were
somewhat less impressive than those of 2a. However, 5a was able to flatten the morphine dose–response curve
(up to 1000 mg/kg morphine) in the TW assay after 30 min of pretreatment,
again suggestive of irreversible-like effects (Supporting Information). The cinnamoylaminomethyl oripavine
derivatives (5b–e) related to 5a all lacked the latter’s opioid receptor partial
agonist character, being opioid receptor antagonists in vivo.11 However, in GPI the p-chlorocinnamoylaminomethyl
derivative (5c) and the p-nitrocinnamoylaminomethyl
derivative (5e) like 5a had partial opioid
receptor agonist activity. 5a and the cinnamoylamino
analogues (5b–e) were all opioid
receptor antagonists in MVD, and it can be concluded that they all
have predominant MOR antagonist activity of pseudoirreversible nature.18

The other fumaroylamino derivative (4a) was also part
of an oripavine structure, but the pharmacophore was directly attached
to the bridged C-ring rather than to a methylene spacer as in 5a. It profiled as an opioid receptor antagonist without agonist
actions both in vitro and in vivo. It had a substantial, long-duration
morphine antagonist effect in TW and AW, but this did not appear to
be pseudoirreversible. The MOR antagonist activity of 4a was similar to that of the p-methylcinnamoylamino
analogue (4d), but 4d had significant opioid
receptor agonist activity in AW. Only the p-nitro
analogue (4e) shared 4a’s lack of
opioid receptor agonist activity, but it had even less MOR antagonist
activity.18

It is of interest to
compare the profile of 4a with
that of the 6,14-etheno analogue (12), designated NIH
10236, reported by Rothman et al.1912 was found to have wash resistant in vitro binding in rat
brain membranes to MOR and DOR, whereas these receptors and KOR were
“alkylated” when equivalent studies were carried out
in vivo with intracerebroventricular administration of 12. The authors concluded that “multiple factors complicate
the use of alkylating agents for in vivo selectivity studies”.
Our studies with 4a suggest that it is a poor alkylating
agent and thus a poor irreversible antagonist.

The action of 1a as an irreversible MOR antagonist
can be attributed to the fumaroylamino group acting as a Michael acceptor
which in physiological conditions reacts with a sufficiently reactive
nucleophile of the MOR, forming a covalent bond.20 It is noteworthy that molecular modeling indicates that
the double bond of the fumaroylamino group in 2a and 5a is conjugated to either the amide or ester functionality
(dihedral angle near 0° or 180°), whereas there is a lack
of conjugation to either in 4a.21 This might explain the lack of irreversible characteristics displayed
by 4a in vivo, as lack of reactivity would prevent Michael
addition and hence covalent bond formation. Recently the crystal structure
of the MOR bound to 1a has been reported.22 We have investigated whether the current ligands (2a, 4a, 5a) can overlay the structure
of 1a in the binding pocket while also interacting with
the nucleophilic residue (Lys233) that 1a covalently
binds to. The results suggest that none of the current series appear
to be able to interact with this or other lysine residues without
adopting a very different binding conformation to 1a,
and so it is not clear that they would interact with the receptor
in an analogous fashion to 1a.

The remaining new
14-substituted derivatives (3a, 6) were
synthesized for comparison with the 14-fumaroylamino
derivative (2a). The 14-dihydrofumaroylamino derivative
(6) had a substantially lower level of antinociceptive
activity than 2a. It had only reversible MOR antagonist
activity in TW, but it was a low potency antinociceptive agent in
AW. The lack of irreversible antagonist activity for the dihydrofumaroylamino
derivative supports the view that the irreversible antagonist activity
of 14-fumaroylaminodihydromorphinone (2a) is the result
of covalent binding to MOR via Michael addition to the fumaroylamino
group.21 The 14-fumaroyloxy derivative
(3a) had weak KOR partial agonist activity in GPI but
no antinociceptive activity in TW or AW. It was a powerful morphine
antagonist in TW but of relatively short duration. Thus, the fumaroyloxy
pharmacophore of 3a failed to match either the MOR agonist
or MOR antagonist profile of the fumaroylamino group in 2a. 3a can also be compared to the 14-cinnamoyloxy derivatives
(3b–d).23 The latter group had substantially higher potency in MVD as opioid
receptor antagonists than 3a (e.g., 3b in
Table 2). In fact, the MOR profile of 3a is somewhat similar to that of its parent, naltrexone,
i.e., a potent reversible MOR antagonist; it is possible that 3a is metabolized in vivo to naltrexone.

In other studies
short chain ester groups have been substituted
for lipophilic aryl groups in active molecules in order to reduce
duration of action, since they offer similar levels of lipophilicity
but are more easily metabolized in vivo.24 Comparison of fumaroylamino derivatives with equivalent cinnamoylamino
derivatives can be seen in this light; in the current series there
is no evidence that the presence of a methyl ester moiety leads to
a shortened duration of action. This may be due to inhibition of metabolic
esterase activity by conjugation in the fumaroylamides.

Conclusions

The fumaroylamino derivatives reported
herein have predominant
MOR antagonist activity that in the cases of the 14-substituted morphinone
(2a) and 7-aminomethyl oripavine (5a) has
irreversible character like the equivalent cinnamoylamino derivatives
(2b–e, 5b–e). The present study confirms the general similarity of the
effects of the two pharmacophores, but the particular substituent
in the cinnamoylamino group offering the closest similarity to the
fumaroylamino derivative varies between series and in particular there
seems to be a greater level of in vivo MOR agonist activity in the
fumaroylamino series. Comparison of 2a and 5a with the prototype fumaroylamino opioid β-FNA (1a) shows that the new ligands have similar MOR irreversible antagonism.
However, like β-FNA they have shorter duration agonist effects,
but the agonism of 2a is predominantly mediated by the
MOR and the agonism of 5a is less clearly defined whereas
β-FNA’s agonist effects are clearly KOR-mediated. The
profile of 2a, MOR agonist activity followed by irreversible
antagonism, might have made it of interest in the search for alternatives
to buprenorphine. However, 2a’s MOR effects compare
unfavorably with buprenorphine’s, particularly the agonist
effects. The MOR agonist activity of 2a is of shorter
duration and of lower efficacy and potency. The MOR irreversible antagonism
of 2a matches that of buprenorphine but again of lower
potency.

Experimental Section

Column chromatography was performed
under gravity over silica gel
60 (35–70 μm) purchased from Merck. Analytical TLC was
performed using aluminum-backed plates coated with Kieselgel 60 F254 from Merck. The chromatograms were visualized using UV
light (UVGL-58, short wavelength), ninhydrin (acidic), or potassium
permanganate (basic). Melting points were carried out using a Reichert-Jung
Thermo Galen Kopfler block or a Gallenkamp MFB-595 melting point apparatus
and are uncorrected. High and low resolution electron impact (EI)
mass spectra were recorded using EI ionization at 70 eV, on a VG AutoSpec
instrument equipped with a Fisons autosampler. 1H NMR and 13C NMR spectra were recorded using a JEOL 270 (operating at
270 MHz for 1H and 67.8 MHz for 13C) spectrometer.
Chemical shifts (δ) are measured in ppm. Spectra were referenced
internally using TMS as the standard. Only diagnostic peaks have been
quoted for proton NMR. Microanalysis was performed with a Perkin-Elmer
240C analyzer. Infrared spectroscopy was performed on a Perkin-Elmer
782 instrument. Chemicals and solvents were purchased from Aldrich
Chemical Company. Compounds were submitted for testing as their oxalate
salts, formed by adding 1 equiv of oxalic acid to an ethanolic solution
of the ligand. Ligands were >95% pure by microanalysis.

Full experimental procedures
and characterization data and figure showing antagonist activity of 5a in TW assay. This material is available free of charge
via the Internet at http://pubs.acs.org.

Supplementary Material

Notes

The authors
declare no
competing financial interest.

Acknowledgments

This work was funded through NIDA Grants DA00254 and DA07315
and the in vitro characterization of compounds carried out through
the NIDA Abuse Treatment Discovery Program (ATDP). Part of the in
vivo evaluation was supported by NIDA Contract No. 7-8859. M.P.T.
was supported by the Wellcome Trust (Programme Grant 082837 to B.
V. L. Potter, University of Bath, U.K.).

[TableWrap ID: tbl3] Table 3
Percent Inhibitiona by New Ligands of the Effect of an ED100 Dose
of Selective Agonistsb in AW

% inhibition

ligand

morphine

BW373U86

bremazocine

2a

62

31

5

3a

10

15

30

4a

70

28

20

5a

95

10

25

aDoses of 32 mg/kg of test ligands
administered 24 h earlier.

bThe agonists used were morphine
(MOR), BW373U86 (DOR), and bremazocine (KOR). Buprenorphine is a full
agonist in the AW test with an EC50 of 0.11 (0.04–0.29)
nM (Jiminez-Gomez and Traynor, unpublished).